探討細胞形狀對於單股核酸包覆奈米粒子的基因轉殖效率

Investigating the Cell Shape on Gene Transfection Efficiency of Oligonucleotide-assembled Nanocomplexes

黃匯雯(Hui-Wen Huang)

指導教授 ： 楊台鴻

國立臺灣大學/工學院/醫學工程學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01904

摘要

細胞形態對於細胞具有很深遠的影響，包括：細胞的增生、分化、細胞骨架的重組和基因表現。目前對於細胞形狀的研究，多專注在改變形狀對於細胞有哪些基因表達的改變，鮮少關注改變細胞形狀與基因轉殖之間的關係。
本研究利用不同培養基材 chitosan 和 TCPS 培養細胞，可成功改變細胞形狀。確立細胞形狀改變後，使用兩種基因轉殖載體，第一種是 ExGEN500 和Plasmid DNA 混和的基因轉殖載體，第二種是在其外包覆單股核酸的基因轉殖載體。研究結果發現，兩種基因轉殖載體於 chitosan 上，細胞的轉殖效率均差於 TCPS 。因此，對細胞進行吞噬實驗，發現細胞吞噬基因轉殖載體在chitosan 和 TCPS 差異不大。但是，於共軛焦顯微鏡的觀察中，發現基因轉殖載體在 chitosan 上，較不容易進入細胞核。因此，可能是細胞降低轉殖效率的原因。
第二部分研究探討，細胞先吞噬基因轉殖載體於 TCPS上，再使用 trypsin 劇烈改變細胞骨架後，重新貼附於 TCPS 或是懸浮於 chitosan 上。發現DNA/PEI轉殖載體的轉殖效率，於短時間吞噬和短時間培養的條件下，有表現增加的趨勢，而長時間吞噬與培養則無此現象，因此推測，改變細胞形狀於吞噬後短時間內，可使基因轉殖蛋白提早表現。
總結兩部分實驗，可發現細胞形狀對於基因轉殖具有很深遠的影響，若是先改變細胞形狀，再進行基因轉殖，其轉殖效率會下降。但先進行基因轉殖，使細胞吞噬基因轉殖載體一段時間後，再改變細胞形狀，其轉殖效率並不會下降太多。顯示若是轉殖粒子進入細胞核內後，改變細胞形狀對於轉殖蛋白的表現不會影響太多。因此，轉殖載體能否進入細胞核是基因轉殖的重要關鍵。

關鍵字

細胞形態 ； 基因轉殖 ； 幾丁聚醣 ； 單股核酸 ； 聚乙烯亞胺

並列摘要

Cell morphology has a profound effect on a range of cellular events, such as proliferation, differentiation, cytoskeletal organization, or presumably gene expression. Currently, regarding the research of cell shapes, the majority focuses on the changes to gene expression after the shape of the cell has been changed, while only a few focus on the relationship between changing the shape of the cell and the effects it has on gene transfection.
This research uses two different materials, chitosan and TCPS, to culture the Hela cells and successfully change the cell shape. Once the cell shape is changed, two gene transfection vectors were used, the first type being a ExGEN500 and Plasmid DNA complex, and the second type being an oligonucleotide coating on the first type complex. According to the results of this research, the cells cultured on chitosan, both gene transfection vectors are less efficient than the cells cultured on TCPS. On the other hand, from the results of the cell uptake experiment, it is observed that there is no difference between the cell uptake of the complexes with chitosan and the complexes in TCPS. However, from the data of the confocal microscopy, DNA/PEI complexes do not transfect into the cell nucleus when cell culture on chitosan. This may be the reason for the lowered gene transfection efficiency.
In the second part of the research, the gene transfection vectors are first uptaken by cell on TCPS, and then, after the use of trypsin to dramatically change the skeleton of the cells, the cells are readhered to TCPS or suspended above chitosan. From this, it is observed that, under a shortly uptaking time and a shortly culturing period, the gene transfection efficiency of DNA/PEI complex is increased, while under a long uptaking time and long culturing period there is no difference in effect. From this, it can be predicted that changing the form of the cell in the shortly uptaking time period after cell uptake can cause gene transfection proteins to perform earlier.
From the two parts of the experiment, it can be seen that the structure of the cell has a large impact on gene transfection, but if the cell strcture is changed first, and then the cell is subjected to gene transfection, the efficiency of the transfection will be decreased. However, if the cell is first subjected to gene transfection, and the gene transfection complex is subjected to cell uptake for a brief period before the cell structure is changed, the efficiency of the gene transfection will not be decreased to the same extent. Also, the research shows that once the gene transfection particles enter the nucleus of the cell, with regards to the transfection protein, changing the structure of the cell will not cause a large effect on performance. Thus, whether the complexes can enter the cell or not is the key to gene transfection.

並列關鍵字

cell morphology ； gene transfection ； chitosan ； oligonucleotides ； polyethyleneimine

參考文獻

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